RBC transfusion is the single most common therapeutic modality given to patients in America, with approximately 1 out of every 70 Americans being transfused each year. Some chronically transfused patients become alloimmunized against many of the over 340 RBC alloantigens, leading to morbidity and mortality due to lack of sufficient compatible RBC units. In addition, RBC alloantibodies can lead to hemolytic disease of the fetus and newborn. Thus, issues of RBC alloimmunization are of high significance to a large number of patients. Antibodies have traditionally been considered part of the adaptive immune system, which forms a response only after exposure to antigen; however, in recent years a new class of pre-existing antibodies (naturally occurring antibodies (nAbs)) have been described that bridge the gap between innate and adaptive immunity. Most nAbs are IgM (nIgMs) that have a limited range of specificities, including autoantigenic determinants on damaged tissues. All units of RBCs that are transfused are stored first, resulting in characteristic patterns of cell damage, including expression of known nIgMs targets. In animal models, we have reported that storage of RBCs increases RBC alloimmunization upon transfusion, a finding that has been observed in some follow up human studies. In this context, this applicaiton will test the hypothesis that nIgMs, pre-existing in nave animals, are involved in increased immunogenicity of stored RBCs through binding conserved motifs that are increased on the RBC surface of stored RBCs, leading to altered antigen presentation, T cell activation and B cell maturation. In addition to nIgMs, normal adaptive IgG to RBC alloantigens can be detected as early as 7-12 days; however, transfused RBCs continue to circulate up to 100- 120 days in humans (50 days in mice). Thus adaptive IgGs can bind to circulating RBCs during a primary immune response, with the potential to affect ongoing RBC alloimmunization. Indeed, we present novel data that anti-RBC IgG, has regulatory effects upon ongoing RBC alloimmunization. In this context, the proposed studies will also test the hypothesis that adaptive anti-RBC IgGs affect ongoing alloimmunization by shuttling antigen to more immunogenic antigen-presenting cells, activation of the antigen-presenting cells, and increased subsequent immunity. These two hypotheses are investigated in the context of 3 specific aims: Specific aim 1: Determine the role of nIgM in initiating adaptive alloimmunity to RBC transfusion Specific aim 2: Mechanisms by which anti-RBC antibodies alter adaptive humoral alloimmunization. Specific aim 3: Test the effects of anti-RBC alloantibodies upon ongoing RBC alloimmunization in a humanized mouse model. These aims use innovative approaches to test mechanistic biology, at both the cellular and molecular level, and also generate an initial bridge to translation into human studies in aim 3.